Project Summary/Abstract Immune system proteins are currently studied with inappropriate methods that fail to account for native diversity present at the cell surface. Indeed, post-translational modifications are often-neglected features that wildly change affinity, depending on the location and composition of the modifications. Thus, it is presently unclear how to appropriately target immune receptors and treat disease or how to reprogram the expressing leukocyte to achieve a desired therapeutic response. Here we propose experiments to completely characterize the modifications, primarily complex carbohydrates, at each amino acid of Fc g receptor III / CD16 in unprecedented detail. We will isolate CD16 from a single cell type from a single donor, covering four biological sources of CD16a. By studying samples from 30 donors, we will create a library of data regarding how each donor modifies CD16a at each site from each cell type. Fc g receptor III / CD16 is the primary receptor that activates antibody-dependent cell- mediated cytotoxicity and contributes to other protective immune responses. Most monoclonal antibody therapies require CD16 binding for efficacy, and antibodies with greater affinity for CD16 provide greater benefit. Therefore, increasing the affinity of the CD16 / antibody interaction will enhance patient outcome. Contemporary efforts have focused exclusively on designing monoclonal antibodies with greater CD16 binding, but our laboratory discovered that variability in Golgi-mediated CD16 processing has a greater impact on antibody binding than antibody processing. We will test the hypothesis that CD16 from natural killer (NK) cells displays smaller and less elaborate post-translational modifications causing NK cells to bind antibody with greater affinity than other CD16+ leukocytes (monocytes and neutrophils) and serum-borne CD16. Though these experiments will analyze multiple types of post-translational modification, our preliminary evidence indicates that asparagine-linked carbohydrates (N-glycans) are the predominant factor that influence antibody binding by CD16. Our lab recently determined that human CD16a with minimally-processed oligomannose-type N-glycans bound 40-fold tighter to immunoglobulin G1 in vitro than CD16a with highly-processed complex-type N-glycans. To test the hypothesis, we will purify CD16 from primary human NK cells, monocytes and neutrophils as well as CD16 from serum to analyze the post-translational modifications with amino acid site- specific resolution. Finally, we will evaluate how CD16a N-glycan processing impacts antibody binding in vitro and with cell activation experiments.